Identification of functional PGH2/TxA2 receptors on human endothelial cells.

Although functional receptors for thromboxane A2 and prostaglandin H2 (TxA2/PGH2) have been identified in platelets and vascular smooth muscle cells, receptor-mediated events in human endothelial cells stimulated by these endoperoxides have not been shown. Using cultured endothelial cells harvested from human umbilical or saphenous veins, we measured the effect of the TxA2 mimetic U46619 on mobilization of cytoplasmic calcium ([Ca2+]i), as well as release of prostacyclin and expression of the proto-oncogene c-fos, intracellular events that have been linked to [Ca2+]i rise in stimulated endothelial cells. Addition of U46619 to confluent fura 2-loaded endothelial cells caused a concentration-dependent rise in intracellular [Ca2+]i, with agonist concentrations of 300 nM producing a maximal [Ca2+]i rise. This [Ca2+]i rise was a uniform response observed in all individual endothelial cells throughout the monolayer, as shown by microspectrofluorimetric visualization. Similar effects were seen with a structurally dissimilar endoperoxide analogue, I-BOP, and with the naturally occurring endoperoxide PGH2. The initial [Ca2+]i rise was not reduced when extracellular [Ca2+]i was chelated with EGTA, but a later "plateau" phase was eliminated. An antagonist of the receptor for TxA2/PGH2 (SQ29548) strongly inhibited [Ca2+]i mobilization. Stimulation of endothelial cells with U46619 also transiently increased expression of the proto-oncogene c-fos, as determined by RNA hybridization, and induced a fivefold increase in prostacyclin release. Thus, endoperoxides can stimulate human venous endothelial cells by means of TxA2/PGH2 receptors, whose occupancy can activate intracellular events associated with functional changes.

Cyanide prevents the inhibition of platelet aggregation by nitroprusside, hydroxylamine and azide.

Sodium cyanide (CN-) in concentrations of 10 uM or more prevented the inhibition of epinephrine (2.5 uM) and of ADP (4.0 uM) induced primary and secondary aggregation brought about by 10 uM sodium nitroprusside (SNP). Cyanide alone in the same concentration had no effect on platelet aggregation induced by epinephrine or ADP. Even when the addition of CN- was delayed for as long as 9 min after epinephrine and SNP, it immediately reversed the SNP block and initiated a bimodal wave of aggregation. The effect of CN- on SNP inhibition of platelet aggregation appears to be competitive and reversible. Although they are less potent inhibitors of platelet aggregation than SNP, the effects of hydroxylamine (HA) and azide were also prevented by SNP. In our hands, sodium nitrite did not inhibit platelet aggregation consistently. The inhibitory effects of glyceryl trinitrate, papaverine and nitric oxide hemoglobin on platelet aggregation were not prevented by CN-. These interactions probably have no significance in vivo, but they indicate that SNP, HA and azide act on platelets and on vascular smooth muscle by similar or identical biochemical mechanisms. They also suggest that there are at least two sub-classes of so-called nitric oxide vasodilators. The effect of CN- may be mediated through an inhibition of the formation of nitric oxide from SNP, HA and azide.